This project involves understanding how changes in Escherichia coli gene expression are coordinated when cell growth is limited by nutrient availability. We are interested in signals provoking changes in cellular levels of a class of regulatory nucleotide analogs of GDP and GTP that bear pyrophosphate residues on the ribose 3' hydroxyl; abbreviated as ppGpp and pppGpp, respectively, or (p)ppGpp collectively. Changes in (p)ppGpp levels are of interest because they are correlated with complex changes in gene expression triggered by sensing starvation for sources of energy, of amino acids or of phosphate. Since (p)ppGpp is not essential for growth, it can be argued that these nucleotides provide predominately regulatory functions for the cell. We reported that (p)ppGpp induces expression of the alternative RNA polymerase sigma factor, sigma-S, encoded by the rpoS gene. This is significant because sigma-S accumulation triggers entry into a globally altered pattern of gene expression characteristic of stationary phase. We now find that (p)ppGpp induction of rpoS occurs at the translational level, a distinctly new level of regulation for (p)ppGpp; (p)ppGpp typically affects transcription. (p)ppGpp-independent mutants of the "housekeeping" sigma factor, encoded by rpoD, have been shown to affect patterns of abortive RNA chain release during in vitro transcription initiation, implicating a promoter clearance mechanism. We continue to explore regulatory relations described between (p)ppGpp, phosphate availability, and polyphosphate (Pn). The functional and sequence relatedness between the pppGpp gamma phosphohydrolase, encoded by gppA, and the major Pn phosphatase, encoded by ppx, has been extended to include preliminary evidence of regulatory parallels as well. Phosphate limitation provokes well known global changes in gene expression owing to activation of a DNA binding protein (PhoB) by phosphorylation as well as (p)ppGpp induction; leading to induction of rpoS. We now find that mutants eliminating either (p)ppGpp or eliminating rpoS alter Pho regulon activity. We have indications that a stationary phase-specific Pn phosphatase may exist in addition to the ppx encoded enzyme expressed during growth. The operon containing ppk and ppx so far known to be involved in Pn synthesis and degradation respectively, has been characterized by the Kornberg laboratory. We have constructed ppkx deletions and shown them to be lethal when combined with a deletion of rpoS. We suspect the rpoS governs stationary phase sources of Pn synthesis and ppkx governs sources of Pn synthesis during exponential growth; Pn may be an essential cell component. To test this, we are collaborating with the Kornberg laboratory, experts in biochemical characterization of Pn synthesis and degradation activities. We are characterizing putative ATP-dependent RNA helicase genes; at least five such "DEAD box" genes exist in E. coli and multiple genes are found in all organisms tested. Deletion analysis gives us the first indication that such genes may provide essential functions.